The effective detection and diagnosis of cancer at an early stage in its development may be critical in the ultimate successful treatment of the disease. It is further extremely important that the diagnosis accurately pinpoint the type or location of the tumor since different tumor types may frequently require the use of different chemotherapeutic or treatment regimens. Finally, it is also necessary that the testing procedure used in diagnosis be precise to such an extent that there is minimal danger that either false positives or false negatives will produce an inaccurate diagnosis.
In this connection, the various procedures coming under the heading of "immunoassay" have shown particular promise in development of tumor specific diagnostic testing. Immunoassay relies, in principle, on the natural reactions of the body's immune system to the presence of foreign substances introduced into the body. The immune system is provoked by these foreign materials, for example, infectious organisms such as bacteria or viruses, to produce antibodies which react specifically with the foreign substance (or antigen) and which, if effective, aid in the elimination of the organisms or foreign matter from the body. The production of antibodies, of course, is not limited to the presence of infectious microorganisms but is also observed in response to many materials which are not normally found in circulation in the body such as cancerous cells, foreign blood group antigens, or fetus-specific antigen.
The relative specificity of antibody for a particular antigen has provided the basis for highly specific and accurate diagnostic testing for various physiological conditions such as infectious diseases, specific tumors, pregnancy and presence of drugs in the body. In practice, the test operates by exposing a test sample suspected of containing a specific antigen associated with a particular disease condition, or antibodies to a particular microorganism, such as the AIDS virus, to a detectably labelled corresponding "immunological partner," i.e., the corresponding antibody or antigen. Alternately, in a "sandwich" type of assay, a test sample containing the antigen to be detected is added to a corresponding antibody, and this is followed by addition of a second labelled antibody to the antigen, providing a detectably labelled "sandwich," indicating the antigen's presence. Competitive binding assays also exist, in which the relative amount of binding of an antigen mimic to an antigen-specific antibody in the test sample is used to indicate the relative amount of antigen present in the sample. In all these immunoassays, a reaction between antigen and antibody indicates the presence of the suspected condition, the reaction being made visibly detectable by the presence of a label on the antigen or antibody. The most frequently used labels are enzymes, to which a substrate is added, causing a catalytic reaction producing a color change; also commonly used are fluorescent, chemiluminescent or radioactive molecules. A wide range of variations on immunoassay techniques are currently available as can be seen, for example, by reference to U.S. Pat. Nos. 4,016,043; 4,424,279 and 4,018,653.
The successful development of an accurate immunoassay for disease detection, of course, requires that an appropriate antigen or antibody is available with which to conduct the assay. For example, the ideal antigen is one which is highly specific to the particular condition of interest, the identification of which, in the test sample, will definitively show the presence of the causative agent or other associated factor. Such antigens would include, for example, a lipopolysaccharide peculiar to a particular species of bacterium; a glycoprotein only found in the coat of a specific type of virus; or cell surface antigens only found in abnormal cell types such as tumor cells. The selection of the appropriate antigen is critical to the accuracy of any diagnosis since selection of an antigen which is not specific to a particular cell type or species may result in a large number of false positive reactions by identification of unrelated cells or organisms also carrying the antigen.
A particular effort has been made to identify tumor cell antigens as markers for specific types of tumors. Developments of a test which can identify a disease connected antigen with specificity will be of substantial value to the clinician not only in early diagnosis, but also in evaluating the progression of the disease and determining effectiveness of ongoing therapy. Some success has been achieved in the development of immunoassays for tumor specific antigens. For example, human chorionogonadotropin (hCG) has been effectively applied to the monitoring of trophoblastic disease. Similarly, certain other antigens, such as alphafetoprotein (AFP), prostatic acid phosphatase (PAP) and carcinoembryonic antigen (CEA), have all been effectively been employed in detection and monitoring of tumors in general and in particular connection with testicular, prostate and colorectal carcinomas. However, outside the aforementioned collection of substances, there has been a relative dearth of other antigens which are truly useful in accurate diagnosis and monitoring; this has been proven particularly true with respect to gynecologic malignancies, especially ovarian carcinomas, which have frequently already spread throughout the pelvic cavity before diagnosis of the condition. Many of these carcinomas typically exhibit a very agressive growth pattern and generally respond well to chemotherapy. Thus, an accurate method by which early diagnosis could be obtained in these diseases is highly desirable.
A substantial amount of research has been devoted to isolating antigens which may be useful in the selective detection of ovarian carcinomas. For example, Knauf and Urback (Am. J. Obstet. Gynecol. 138: 1222, 1980; Cancer Res. 41: 1351, 1981) have described an antigen named OCA which is significantly elevated in the plasma of 76% of patients with ovarian cancer; unfortunately, the antigen also occurs at high levels in about 10% of patients with benign gynecologic disorders, pregnant females and disease free controls, thus potentially rendering a positive test result unreliable. Similarly, Smith and Ol (Obstet. & Gynecol. Surv. 39: 346, 1984) report an ovarian cancer associated antigen, but this antigen has not yet been evaluated as to its specificity and suitability in diagnosis or monitoring of afflicted patients.
A recent discovery by Bast, et al. (N. Engl. J. Med. 309: 883 (1983) of a serous cystadinocarcinoma ovarian antigen, known as CA125, has proven to be of significant value in monitoring patients with ovarian cancer. This antigen was isolated by using a monoclonal antibody, OC125, raised by stimulation of mice with ovarian cancer cell line OVCA 433. It has been shown to recognize cell surface antigens of the OVCA 433 cell as well as 13 of 14 other ovarian cancer cell lines and a melanoma cell line. The antigen is a high molecular weight (&gt;200,000 daltons) glycoprotein which has been partially purified from tissue culture medium (Masuko, et al., Cancer Res. 44: 2813, 1984). With use of the aforementioned monoclonal antibody, sera of several patients with various types of cancer was tested for the presence of CA125. Results showed that 83% of patients with ovarian cancer had elevated levels (&gt;35 units/ml), whereas only 1% of 888 normal patients sera showed titers above this level. Although data indicate a certain level of non-ovarian specificity of the CA125 antigen, the observation of rising and falling levels of antigen with progression and regression of the disease in patients with ovarian cancer show utility of the antigen in monitoring the progress of the disease in already diagnosed patients. The use of CA125 for diagnostic purposes is further complicated by the fact it appears to be, to some extent, a normal product of development. Significant quantities have been found in amniotic fluid during gestation (O'Brien, et al., Soc. Gyn Invest. Abstract, p. 54, March, 1985. It has also been found to increase in connection with benign disease conditions such as endometriosis or pelvic inflammatory diseases. The existence of even small amounts of CA125 in normal tissue provides a significant chance of cross reactivity of the CA125-specific antibodies with non-tumor tissues as well as with cancerous tissues. Therefore, again, although there is substantial promise in the CA125 antigen as a potential marker for ovarian cancer, there has not yet been determined a method by which interference from non-ovarian tumor and normal tissue levels of CA125 can be eliminated. Further, the known monoclonal antibody OC125 appears to react with both the normal and tumor antigen. Therefore, there still does not exist a truly ovarian tumor specific immunoassay which can reliably be used to detect and monitor the ovarian carcinomas without the possibility of cross-reaction with normal tissue. It has now been unexpectedly discovered that the antigen CA125 contains a heretofore undiscovered subunit which appears to be specific to ovarian tumor-associated CA125. Normal tissue tested has failed to show the presence of this fraction of the CA125 molecule. Thus, the discovery of this unique subunit now presents a means by which the testing procedure utilizing CA125 as a marker for ovarian cancer can be refined to reduce or eliminate cross reaction with non-ovarian tumor associated CA125 antigen.